Microbiology is an important,fundamental and obligatory course in contemporary life science.This article introduces that teaching group of microbiology in Nankai University realizes transformation of teaching center,fully embodies the modernization of teaching notion and gives full play to students' main effect practically by adhering to teaching reform as center,optimizing teaching method as measure,communicating in and after class and using multi-media and teaching web.Therefore,teaching system is established to adapt to modern teaching notion and eventually microbiology course becomes a cultivation platform to foster elites with both solid fundamental theory and innovating mind.

Genomic DNA of two fungi Thamnidium elegans and Umbelopsis isabellina were extracted with an amended Cetyltrimethyl Ammonium Bromide (CTAB) method. This modified method uses repeated freezing in liquid nitrogen and thawing with combination of shocking with glass beads to replace of the tra- ditional method. Quality and concentration of DNA extracted by the modified methodwere tested. Compared with the traditional method, higher yield and purity of genomic DNA were obtained with less amount ofmy- celium. The result indicted that this is a simple and highly efficient method, which is suitable to treat many samples at one time and for basic molecular experiments, such as restriction endonuclease reaction and PCR.

OBJECTIVEThe purpose of this study was to observe the tissue tolerance of Ti-HA functionally graded-material (FGM) and the form of the material-bone interface.

METHODSThe sintered Ti -HA FGM, pure HA and pure Ti were respectively implanted into the parietal bone of rabbits. The specimens were observed by SEM at 2, 4, 8 postoperative weeks.

RESULTSIn the early stage, the new bone surrounding the Ti -HA FGM formed earlier with larger amount and better maturity than the pure Ti. The condition was similar to the pure HA. Two months after the operation, direct bonding of material-bone interface was formed between the Ti -HA FGM and the new bone as an integral body. However, there was a little space left between the new bone and the pure Ti.

CONCLUSIONSThe Ti -HA FGM has good tissue tolerance. Its early integration with bone is similar to pure HA and better than pure Ti.

Animals ; Bone Substitutes ; chemistry ; Female ; Hydroxyapatites ; chemistry ; Male ; Materials Testing ; Microscopy, Electron, Scanning ; Osseointegration ; Rabbits ; Titanium ; chemistry

Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase.
Agrobacterium tumefaciens ; genetics ; Fatty Acid Desaturases ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mortierella ; enzymology ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Polymerase Chain Reaction ; Tobacco ; genetics ; metabolism ; gamma-Linolenic Acid ; biosynthesis

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.
Cloning, Molecular ; Fatty Acids ; analysis ; Gas Chromatography-Mass Spectrometry ; Linoleoyl-CoA Desaturase ; genetics ; Mortierella ; enzymology ; genetics ; Pichia ; genetics ; Plasmids ; gamma-Linolenic Acid ; analysis

Delta6-fatty acid desaturase is a membrane-bound enzyme, which is rate-limiting for the biosynthesis of polyunsaturated fatty acids. A cDNA sequence putatively encoding a delta6-fatty acid desaturase was isolated from Rhizopus arrhizus NK300037 using RT-PCR and RACE methods in our previous work. Sequence and function analysis indicated that this sequence was a novel delta6-fatty acid desaturase gene which had an open reading frame of 1377bp coding 458 amino acids of 52kD. The methylotrophic yeast Pichia pastoris, has been developed into a highly successful system for the production of a variety of heterologous proteins during the past 20 years. In this work, the Rhizopus arrhizus delta6-fatty acid desaturase gene (RAD6) was subcloned into expression vector pPIC3.5K to generate a recombinant plasmid pPICRAD6, which was subsequently transformed into Pichia pastoris strain GS115 for heterologous expression by electroporation method. Total fatty acids were extracted from the induced cells and methylated. The resultant fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Fatty acids analysis showed that the coding product introduced a new double bond at delta6 position of appropriate fatty acid substrates including C16:1, C17:1, C18:1, linoleic acid and alpha-linolenic acid without chain length specificity of fatty acids. Furthermore, modification of sequence flanking AUG codon of this delta6-fatty acid desaturase gene increased the expression of target gene in P. pastoris. All of these results suggest that P. pastoris is an optimal expression system of delta6-fatty acid desaturase gene.
Cloning, Molecular ; Electroporation ; Fungal Proteins ; biosynthesis ; genetics ; Genetic Vectors ; Linoleic Acid ; metabolism ; Linoleoyl-CoA Desaturase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Rhizopus ; enzymology ; genetics

Adolescent ; Adult ; Analysis of Variance ; China ; Cross-Sectional Studies ; Homosexuality, Male ; statistics & numerical data ; Humans ; Internet ; statistics & numerical data ; Male ; Middle Aged ; Surveys and Questionnaires ; Young Adult

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.
Cloning, Molecular ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Engineering ; methods ; Phylogeny ; gamma-Linolenic Acid ; biosynthesis

OBJECTIVETo determine the optimal cytokine combinations with hepatic growth factor (HGF) that results in the most significant simultaneous in vitro expansion of cc-kit(+)Lin(-) cells derived from the bone marrow.

METHODSC-kit(+)Lin(-) cells were isolated from mouse bone marrow using a high-gradient magnetic cell sorting system (MACS) and expanded in the presence of stem cell factor (SCF), FLt-3 ligand (FL), leukemia inhibitor factor (LIF) thrombopoietin (TPO) and different concentrations of HGF for 7days in a liquid culture system. The total cell number and Annexin-V-positive cell number were counted, and the antigen expressions were studied with fluorescence-activated cell sorting (FACS).

RESULTSIn each group, c-kit(+)Lin(-) cells were expanded effectively and rapidly by 2 to 8 folds. Addition of 10 ng/ml HGF into SCF+FL+LIF+TPO resulted in the most significant expansion of c-kit(+)Lin(-) and total cells by 8.00 and 45.43 folds, respectively, with cell apoptosis rate of 17.42 %. But as the concentration of HGF increased, the c-kit(+)Lin(-) cells and the apoptosis rate decreased.

CONCLUSIONHGF at10 ng/ml shows optimal synergistic effect with SCF, FL, LIF and TPO in expansion of c-kit(+)Lin(-) cells, and excessive HGF may induce cell differentiation.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Hepatocyte Growth Factor ; pharmacology ; Mice ; Mice, Inbred BALB C ; Proto-Oncogene Proteins c-kit ; metabolism

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.
ADP Ribose Transferases ; metabolism ; secretion ; Bacterial Proteins ; genetics ; metabolism ; secretion ; Bacterial Toxins ; metabolism ; Gene Expression Regulation, Bacterial ; Mutation ; Peptide Hydrolases ; genetics ; metabolism ; Pore Forming Cytotoxic Proteins ; genetics ; secretion ; Protease Inhibitors ; pharmacology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity